Sepsis, a dysregulated systemic response to infection, can lead to organ failure. The lung is particularly vulnerable, and acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) can develop early in sepsis. Despite the use of lung-protective ventilation techniques, ALI/ARDS remains a significant clinical challenge associated with high morbidity and mortality [1]. Therefore, there is considerable interest in elucidating the underlying molecular pathways and identifying new therapeutic targets for sepsis-induced ALI.

Inflammatory responses and oxidative stress are complex pathways that characterize the pathophysiology of endothelial barrier dysfunction [2]. It is suggested that the innate immune response is triggered by lipopolysaccharide (LPS) from gram-negative bacteria, acting as an endotoxin and binding to its cell membrane receptor. This exposure to LPS rapidly and robustly generates reactive oxygen species (ROS) and inflammatory factors, disrupting the endothelial barrier and causing microvascular leakage. To prevent and treat sepsis-induced ALI, regulatory molecules that control oxidative stress and the inflammatory response are crucial [3, 4].

Furthermore, studies have shown that ENT reduces NF-kB signaling, which acts as a negative regulator of the toll-like receptor-induced inflammatory response. Previous research has also demonstrated that ENT contributes to the downregulation of mitochondrial reactive oxygen species (ROS) generation [5]. Although these studies shed light on the potential role of ENT in the inflammatory response and oxidative stress, its regulatory role in an experimental model of sepsis-induced ALI remains unclear [6, 7]. In this study, our aim was to explore the function and underlying mechanisms of ENT in the control of sepsis-induced ALI.

For this study, 24 male Sprague-Dawley rats weighing between 180 and 240 g were used. The rats were housed in the animal facility at Al-Zahrawi University College (Iraq), maintained at a temperature of 25\(^\circ\)C with a humidity level of 60-65%, and subjected to a 12-hour light-dark cycle for 24 hours a day. During a three-week acclimatization period, the rats had ad libitum access to food and water. All research was conducted in accordance with the guidelines of the Animal Care and Use Committee of the Faculty of Medicine at Kufa University and complied with the US National Institutes of Health’s Guide for the Care and Use of Laboratory Animals.

Ketamine and xylazine were administered intraperitoneally (i.p.) to the animals at doses of 80-100 mg/kg and 8-10 mg/kg, respectively, to induce general anesthesia [8]. After shaving and cleaning the abdomen with 80% ethanol, either 0.9% NaCl [9].

In brief, rats were anesthetized intraperitoneally (i.p.) with 100 mg/kg ketamine and 10 mg/kg xylazine. An abdominal laparotomy was performed to expose the cecum through a small (approximately 1.5 cm) midline incision. The cecum was ligated just below the ileocecal valve, and it was punctured twice using a 22-gauge needle. After gently manipulating the cecum to release some fecal material, it was reinserted into the abdominal cavity. The abdominal incision was then closed using 5/0 surgical sutures [10]. Rats were monitored every four hours during the first 24 hours and were provided unrestricted access to food and water in their cages thereafter. After 24 hours, the animals were euthanized, and heart and lung samples were collected. The lung tissue was divided into two parts, cleaned with 0.9% saline, and stored in either 0.9% NaCl or 10% formalin at -20\(^o\)C for further examination [11].

Twenty-four rats were randomly assigned to one of four groups (n=6) for the study. The Sham group received intraperitoneal (i.p.) anesthesia during an operative laparotomy but did not undergo cecal ligation and puncture (CLP). The Control group underwent an operative laparotomy with CLP but did not receive any medication. The Vehicle group underwent the same surgical procedure as the Control group but received only the vehicle for etanercept, administered as NaCl i.p. 60 minutes prior to surgery. After euthanizing the animals under anesthesia and extracting the organs, a 24-hour period elapsed [12].

Histopathology

The excised lungs were fixed in 10% formalin before being embedded in paraffin wax. The tissues were sectioned into 5 \(\mu\)m-thick slices and stained with hematoxylin-eosin (H&E) for histological examination. A pathological grading scale was employed in this study, with mild (1), moderate (2), and severe (3) indicating edema, eosinophilic neurons, and necrosis, respectively. Severe (3) represented necrosis, edema, eosinophilic neurons, and red blood cell (RBC) populations. A normal (uninjured) state was defined as the absence of edema, RBCs, and eosinophilic neurons [13].

Immunohistochemistry

Levels of TLR2 and TLR4 were assessed using immunohistochemistry (IHC). Tissues from the treated and untreated groups were processed following the manufacturer’s instructions, and the number of cells labeled with TLR2 and TLR4 antibodies was counted [14]. \[QQ = P \times I,\] where Q is the quick score, P is the percentage of positive cells, and I is the intensity.

ELISA

Modulation of NF-\(\kappa\)B p65: The NF-\(\kappa\)B p65 level in lung tissue from all groups was assessed using the ELISA technique at the conclusion of the experiment.

Statistical Analysis

Statistical analysis was conducted using SPSS version 26. The data were presented as Mean \(\pm\) Standard Error of the Mean (SEM). For multiple group comparisons, ANOVA was employed, followed by a post-hoc test with Bonferroni correction. The Kruskal-Wallis test was used to assess the statistical significance of the mean score for histological alterations in heart tissue. A p-value of 0.05 was considered statistically significant.

Modulation of NF-\(\kappa\)B p65

Our data indicated that both the control and vehicle groups significantly increased NF-\(\kappa\)B p65 levels compared to the sham group (p<0.05). However, NF-\(\kappa\)B p65 levels were significantly lower (p<0.05) in the group treated with etanercept compared to both the control group and the vehicle-treated group. There was no significant difference in NF-\(\kappa\)B p65 levels between the control and etanercept-treated groups (p>0.05) (Table 1 and Figure 1).

Modulation of TLR2 and TLR4

Increased levels of TLR2 and TLR4 were observed in the IHC examination. TLR expression was induced in both the control and vehicle-treated groups but not in the etanercept-treated group or the sham group (Figure 2). In control positive tissue (normal spleen tissue), TLR expression was significantly enhanced (Figure 2).

Histological Evaluation of Lung Tissue Damage

No lung tissue damage (necrosis, edema, dark eosinophilic neuron, or bleeding) was observed in the sham group (Figure 3). In contrast, both the vehicle-treated group (Figure 3) and the control group (Figure 3) exhibited abnormal lung structure characterized by edema, necrosis, dark eosinophilic neurons, and bleeding. Rats treated with etanercept showed reduced lung damage compared to the control and vehicle-treated groups (Figure 3).

When comparing the lung tissues, there was no noticeable difference between the control group and the vehicle group, and the histopathological scores of both groups were significantly increased compared to the sham group \((p>0.05)\). However, in the etanercept-treated group, the histopathological scores were significantly reduced \((p<0.05, 65\%)\) compared to both the control group and the vehicle-treated group. The results indicate no statistically significant difference \((p>0.05)\) between the etanercept-treated group and the sham group.

In our investigation, we found that etanercept (ENT) prevented the development of sepsis-induced acute lung injury (ALI). CLP exacerbated sepsis-induced pulmonary pathology and vascular endothelial hyperpermeability. The effects of CLP on sepsis-induced ALI primarily depend on the regulation of the inflammatory response and oxidative stress.

An excessive and uncontrolled inflammatory response is considered the primary cause of ALI. Extensive lung inflammation leads to basement membrane breakdown and promotes alveolar-capillary membrane permeability [15, 16]. Wolthuis et al. demonstrated that etanercept, a recombinant human soluble TNF receptor fusion protein, reduced neutrophil infiltration and mitigated ventilator-induced lung injury [17].

During our study, CLP significantly increased serum TNF-\(\alpha\) concentration levels over the course of 24 hours. Similar findings have been previously reported, showing that the CLP group exhibited cytokine levels approximately three times higher than the sham group [18, 19]. ENT significantly reduced TNF-\(\alpha\) levels. Our data demonstrate the effective reduction of TNF-\(\alpha\) by ENT, consistent with the mechanism of action of ENT, which inhibits TNF-\(\alpha\) production and renders it physiologically inactive.

The current study suggests that the regulation of pro-inflammatory mediators is essential for ENT to exert its anti-inflammatory effects. Furthermore, ENT treatment appeared to prevent CLP-induced septic lung damage. H&E staining of lung tissues from the CLP model group confirmed the induction of lung damage, as evidenced by tissue necrosis and inflammation. Treatment with ENT effectively ameliorated these effects.

Previous research has shown that ENT reduces sepsis-induced lung damage by inhibiting the NF-\(\kappa\)B signaling pathway. Activation of NF-\(\kappa\)B has been linked to increased production of inflammatory cytokines such as TNF-\(\alpha\), IL-1, and IL-6 in lung cells [20, 21]. Our data indicate that etanercept reduces inflammatory responses following lung injury by suppressing NF-\(\kappa\)B p65 and TLR levels. Moreover, tissue damage resulting from CLP was significantly reduced in the etanercept-treated group. These findings suggest that etanercept can reduce inflammation and improve acute lung injury.

All authors made equal contributions to this research.

The authors declare that they have no conflicts of interest.

This research did not receive any external funding.

Verbal consent has been obtained where necessary.

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